ARTICLE IN PRESS

Systematic and Applied Microbiology 29 (2006) 3–14

www.elsevier.de/syapm

MINIREVIEW
Bacterial tyrosinases
Harald Clausa,, Heinz Deckerb
a
Institute for Microbiology and Wine Research, University of Mainz, Becherweg 15, D-55099 Mainz, Germany
b
Institute for Molecular Biophysics, University of Mainz, Jakob Welder Weg 26, D-55128 Mainz, Germany

Received 21 June 2005

Abstract
Tyrosinases are nearly ubiquitously distributed in all domains of life. They are essential for pigmentation and are
important factors in wound healing and primary immune response. Their active site is characterized by a pair of
antiferromagnetically coupled copper ions, CuA and CuB, which are coordinated by six histidine residues. Such a ‘type
3 copper centre’ is the common feature of tyrosinases, catecholoxidases and haemocycanins. It is also one of several
other copper types found in the multi-copper oxidases (ascorbate oxidase, laccase).
The copper pair of tyrosinases binds one molecule of atmospheric oxygen to catalyse two different kinds of
enzymatic reactions: (1) the ortho-hydroxylation of monophenols (cresolase activity) and (2) the oxidation of
o-diphenols to o-diquinones (catecholase activity). The best-known function is the formation of melanins from
L-tyrosine via L-dihydroxyphenylalanine (L-dopa). The complicated hydroxylation mechanism at the active centre is
still not completely understood, because nothing is known about their tertiary structure. One main reason for this
deficit is that hitherto tyrosinases from eukaryotic sources could not be isolated in sufficient quantities and purities for
detailed structural studies. This is not the case for prokaryotic tyrosinases from different Streptomyces species, having
been intensively characterized genetically and spectroscopically for decades. The Streptomyces tyrosinases are non-
modified monomeric proteins with a low molecular mass of ca. 30 kDa. They are secreted to the surrounding medium,
where they are involved in extracellular melanin production. In the species Streptomyces, the tyrosinase gene is part of
the melC operon. Next to the tyrosinase gene (melC2), this operon contains an additional ORF called melC1, which is
essential for the correct expression of the enzyme.
This review summarizes the present knowledge of bacterial tyrosinases, which are promising models in order to get
more insights in structure, enzymatic reactions and functions of ‘type 3 copper’ proteins in general.
r 2005 Elsevier GmbH. All rights reserved.

Keywords: Tyrosinases; Haemocyanins; ‘Type 3 copper’ proteins; Melanin; Bacteria; Streptomyces

Corresponding author. Tel.: +49 6131 3923547;
fax: +49 6131 3922695.
E-mail addresses: hclaus@uni-mainz.de (H. Claus),
decker@biophysik.biologie.uni-mainz.de (H. Decker).
Introduction
Tyrosinases (E.C. 1.14.18.1) are copper-containing
enzymes which are ubiquitously distributed in nature
[50,60,84]. They are essential for the formation of
melanin [7,65,70] and various other functions. In plants,
sponges and many invertebrates, they are important
components of wound healing and the primary immune

0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.syapm.2005.07.012
 ARTICLE IN PRESS
4 H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14
response [9,62,84]. In arthropods they are also involved
in sclerotization of the cuticle after molting or injury. In
mammals tyrosinases are found in melanocytes of the
retina and skin [28].
Tyrosinases use molecular oxygen to catalyse two
different enzymatic reactions [21,48,50,77,84]: (i) the
ortho-hydroxylation of monophenols to o-diphenols
(monophenolase, cresolase acticity) and (ii) the oxida-
tion of o-diphenols to o-quinones (diphenolase, catecho-
lase activity). The reactive quinones polymerize non-
enzymatically to the macromolecular melanins (Fig. 1).
It should be noted that often the name phenoloxidase is
used in literature, which summarizes tyrosinases, cate-
choloxidases and laccases as well. Because of their
overlapping substrate specificities, a positive test for
catecholase activity does not necessarily mean that the
enzymes exhibit cresolase activity.
In spite of intensive biochemical investigations since
decades, there exist only limited informations about the
protein structure and the exact reaction mechanisms.
Some reasons for these deficits are difficulties in
purification of sufficiently high amounts of tyrosinases
from eukaryotic sources due to low enzyme concentra-
Fig. 1. Enzymatic activities of tyrosinases and related copper
enzymes. Tyrosinases (EC 1.14.18.1) comprise both reactions,
the mono- and diphenolase activity. For the hydroxylation
reaction one atom of O2 is incorporated into the aromatic ring
of the monophenol and the other one is reduced to H2O with
the resulting o-diphenol as the hydrogen donor. In this respect,
tyrosinase is a ‘mixed function oxidase’. The catecholoxidases
(EC 1.10.3.1) exert only the diphenolase activity but not the
chemically fastidious hydroxylase step. Laccases (EC 1.10.3.2)
have no hydroxylase activity but oxidize mono- and diphenols
by a radical mechanism. Tyrosinases, catecholoxidases and
laccases are often summarized as phenoloxidases.
tions, contamination with pigments, occurrence of
isoenzymes or post-translational modifications.
The copper binding sites of tyrosinases share a high
sequence homology with the haemocyanins, the oxygen
carrier proteins of the molluscs and arthropods
[20,22,23,40,84,85]. During evolution, a functional change
of this protein family has been proposed: from enzymatic
oxygen detoxification towards oxygen transport [40].
The common feature is a ‘type 3 copper centre’, a
diamagnetic spin-coupled copper pair [21,48,50,77,84].
Each of the two metal atoms, CuA and CuB, of the
active site are coordinated by three conserved histidines
which are located in a ‘four a-helix bundle’. During the
catalytic cycle the ‘type 3 copper centre’ can adopt
different functional forms: the oxy-state [Cu(II)–O22 –
Cu(II)], deoxy-state [Cu(I) Cu(I) ], half-met state [Cu(I)
Cu(II)] and the met state [Cu(II)–OH –Cu(II)]. In the
latter case the two copper atoms are bridged by hydroxo
ions. The valences of the two copper atoms change from
Cu(I) to Cu(II), which can be followed spectroscopi-
cally. In the oxy-state the molecular oxygen is reversibly
bound as a peroxide between the two copper atoms in a
‘side-on’ conformation (Fig. 5). In the absence of any
substrate more than 85% of the enzyme is in the met
state, which can be regarded as the resting form of
tyrosinase. According to current conceptions, both, the
met- and the oxy-state of tyrosinases enable the
diphenoloxidase activity, whereas the monohydroxylase
reaction requires the oxy-state.
Although much work on bacterial tyrosinases was
published since decades, most studies did not concen-
trate on structural topics. Nevertheless, tyrosinases from
microorganisms may be helpful to get more insight in
the common architecture of these enzymes and espe-
cially in the complicated monohydroxylase reaction
[5,6,79,80,83]. This review will focus on Streptomyces
tyrosinases in the light of recent results on ‘type 3
copper’ proteins in general.
Streptomyces tyrosinases and melanization
Actinomycetes are Gram-positive soil bacteria with
mycelious growth. Members of the genus Streptomyces
are involved in the formation and/or degradation of
complex biopolymers like lignin, melanins, and humic
substances [47]. In addition, they are important industrial
sources of antibiotics and other secondary metabolites.
About 40% of Streptomyces species produce melanin-
like exopigments on tyrosine-containing agar media,
which mostly but not always correlate with the
appearance of an extracellular tyrosinase activity
[12,16]. The typical double enzymatic activity of
tyrosinases could be demonstrated in melanin-positive
species, whereas melanin-negative mutants lost the
 ARTICLE IN PRESS
H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14 5

cresolase activity, but sometimes retained some catecho-
lase activity [12,16]. Tyrosine methylester and caffeic
acid proved the best substrates for measuring both
enzymatic activities of Streptomyces tyrosinase. Electro-
phoretic characterizations revealed that the intra- and
extracellular tyrosinase of one species were identical, but
that the enzymes of different species were not. After
isoelectric focusing, several tyrosinase bands appeared
in some species, indicating the presence of isoenzymes.
The heterogeneity of Streptomyces tyrosinases was also
reflected by different Km values and temperature
stabilities [12,16].
Tyrosinases have been also found in some Gram-
negative bacteria. A thermostable tyrosinase with
maximum activity at 70 1C and pH 9.5 has been isolated
from Thermomicrobium roseum [46]. The active form
seems to be a homodimer of two 43,000 Da subunits.
The intracellular tyrosinase of Marinomonas mediterra-
nea [54,55] is an example for its occurrence in marine
bacteria. Other tyrosinases have been purified from
Vibrio tyrosinaticus [69] and demonstrated in Pseudo-
monas melanogenum decades ago [88].
In contrast to eukaryotic organisms, for bacterial
tyrosinases there are no reports about post-translational
processings, e.g., proteolytic activation of proenzymes
or glycosylations.

Purification
Regulation
The first bacterial tyrosinases have been purified from
cell extracts of Streptomyces nigrifaciens [63] and
For most bacterial tyrosinases it is not known whether
Streptomyces glaucescens [51]. Unlike most eukaryotic
they are produced constitutively or inducibly. Tyrosinase
tyrosinases, the active form of the S. glaucescens protein
synthesis by S. glaucescens is surprisingly not induced by
is a monomer, without tendency of concentration-
tyrosine, but by other amino acids like phenylalanine,
dependent aggregation as shown by analytical ultracen-
methionine and leucine [2]. Methionine is also the
trifugation. The enzyme has a molecular mass of
inducer of the tyrosinase from S. antibioticus [4,41].
29,100 Da in SDS-PAGE and its maximum activity at
The expression of the S. castaneoglobisporus tyrosinase is
pH 6.8. The extracellular tyrosinase of S. glaucescens
favoured by methionine and copper [38]. Otherwise, the
was isolated 10 years later from the culture supernatant
transcription of the S. michiganensis tyrosinase is
[17]. The intra- and extracellular forms were identical in
induced by copper and repressed by ammonium [34].
their molecular masses, N-terminal sequences and
In chemostat experiments, oxygen was found to be a
cresolase/catecholase ratios [17,51]. Bernan et al. [3]
negative regulator of the tyrosinase of S. glaucescens [87].
purified the intra- and extracellular tyrosinase of
The small thermostable tyrosinase from B. thuringiensis
Streptomyces antibioticus. For homologous overexpres-
has been found to be heat-inducible [53].
sion of the enzyme, the mel gene was amplified using the
Recently, the expression of tyrosinase activities and
multi-copy plasmid pIJ702. The molecular mass deter-
melanin formation in the Gram-negative marine bacter-
mined by SDS-PAGE and size-exclusion gel chromato-
ium M. mediterranea has been studied at a molecular
graphy was 29,500 Da. The intra- and extracellular
level [56]. It has been demonstrated that both phenolox-
forms were identical with respect to molecular size and
idases (tyrosinase and laccase) of this species (see
N-termini (as determined by Edman degradation). The
Table 1) are subjected to coordinate regulation. During
extracellular tyrosinase of Streptomyces michiganensis
bacterial growth in a complex medium, the enzymatic
has been isolated from a 10 l fermentation broth [67].
activities are induced in the stationary phase. A sensor
The purified enzyme exhibited two bands corresponding
kinase (PpoS) which has been cloned and sequenced,
to 32,000 and 34,500 Da in SDS-PAGE, however, only
was shown to be involved in up-regulation of both
one band at pH 9.0 after isoelectric focusing. The
enzymatic activities. However, the environmental signals
tyrosinase showed activity with various monophenols
sensed by the PpoS are yet unknown.
(tyrosine, tyrosine-ester, p-coumaric acid) and diphenols
On protein level the tyrosinases from M. mediterranea
(L-dopa, caffeic acid, catechol). The enzyme from
[54] and a B. thuringiensis strain [53] are activated by
Streptomyces castaneoglobisporus has been efficiently
SDS. Similar to haemocyanins, the detergent may induce
overexpressed in Escherichia coli and the his-tagged
conformational changes which result in a better acces-
protein purified on a Ni(II)-bound affinity column [45].
sibility of the substrate to the catalytic site [20,22,23,40].
A heat-inducible tyrosinase from a Bacillus thurin-
giensis strain has been purified by a simple one-step
purification step [53]. With only 14 kDa, this tyrosinase
has one of the lowest molecular masses among the Spectroscopic characteristics
known tyrosinases from different sources. In contrast to
Streptomyces tyrosinase, a dimer is the presumptive Intensive spectroscopic studies have revealed the
active form. surrounding of the two copper ions in tyrosinase.
 ARTICLE IN PRESS
6 H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14

Table 1. Bacterial tyrosinases and helper proteins

Species (abbr.) Protein accession no. or aa MolecularP pIP

reference mass (Da)

Gram-negative bacteria
N
Marinomonas mediterranea (Ma.m.) AAF75831a 675 74,469 4.84
N
Marinomonas mediterannea AAV49996 485 53,089 4.85
N
Marinomonas mediterranea AAV49997b 250 28,567 9.89
N
Nitrosomonas europaea (Ni.e.) NP841294 500 53,929 5.26
N
Rhizobium etli (Rh.e.) NP659960 609 67,418 7.28
N
Sinorhizobium meliloti (Si.m.) P33180 494 54,110 4.65
N
Ralstonia solanacearum (Ra.s.) NP519622 412 43,955 8.44
N
Stenotrophomonas maltophila (St.m.) AAC16658 169 18,644 9.27
Pseudomonas melanogenum (Ps.m.) [88] n.d. n.d. n.d.
Thermomicrobium roseum (Th.r.) [46] n.d. 43,000c 4.9
Vibrio tyrosinaticus (Vi.t.) [69] n.d. 38,500d n.d.
41,000d
Gram-positive bacteria
N
Bacillus cereus (B.ce.) AAP92115 247 28,509 5.47
N
Bacillus thuringiensis (B.th.) AAW29015 151 16,816c 4.87
N
Corynebacterium efficiens (Co.e.) NP738366 415 46,405 5.16
N
Streptomyces antibioticus (S.an.) P07524 272 30,608 7.17
N
P17687b 146 14,884 6.54
N
Streptomyces avermitilis (S.av.) NP828532 299 33,528 9.33
N
NP826538b 128 13,555 6.64
U
Streptomyces castaneoglobisporus (S.ca.) Q83WS2 273 31,038 6.20
U
Q83WS1b 126 13,001 6.42
U
Streptomyces coelicolor (S.co.) Q9KYH2 288 33,104 9.66
U
Q9KYH1b 189 19,364 7.15
U
Streptomyces galbus (S.ga.) P55022 276 31,310 9.33
U
P55046b 126 12,916 6.69
U
Streptomyces glaucescens (S.gl.) P06845 273 30,743 6.40
U
P55047b 134 13,593 8.34
U
Streptomyces griseus (S.gr.) Q9ZN72 306 35,508 8.90
U
Q9ZN73b 127 13,708 11.8
U
Streptomyces lincolnensis (S.li.) P55023 273 30,723 6.84
U
P55048b 140 14,219 7.10
N
Streptomyces lavendulae (S.la.) 2113331B 273 30,989 6.8
N
2113331A 156 16,994 11.9
U
Streptomyces tanashiensis (S.ta.) Q9F1K7 275 31,287 6.84
U
Q9F1K8b 126 12,505 9.39
Streptomyces sp. KY-453 (S.sp.) [89] n.d. 29,000 9.9
Streptomyces michiganenis (S.mi.) [67] n.d. 32,000d 9.0
34,500d
Streptomyces nigrifaciens (S.ni.) [63] n.d. 18,000 n.d.

n.d., not determined.

N
NCBI database; UUniProt database; Pcalculated with the Protparam tool (http://www.expasy.org/tools/protparam.html).
a
Multipotent phenoloxidase.
b
Chaperon-like helper protein.
c
A dimer is the presumptive active form.
d
Isoenzymes.

Electron-spin-resonance (ESR) and Raman spectro-
scopy classify tyrosinase as a coupled binuclear copper
protein (type 3 copper) [50,77,79,80,83]. The paramag-
netic [Cu(I) Cu(II)] half-met state of the dinuclear
copper centre of the tyrosinase from S. antibioticus has
been analysed by pulsed ESR and hyperfine sublevel
correlation spectroscopy in the presence and absence of
inhibitors like p-nitrophenol and L-mimosine [5,6,83].
Comparisons of these inhibitor binding studies with the
known crystal structure of haemocyanin may help in the
future to reconstruct a model structure for the half-met
state of the tyrosinase which would give an explanation
 ARTICLE IN PRESS
H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14
for the binding of mono- and diphenols at the active site.
In addition, 1H NMR measurements in the presence of
substrate analoga will help to understand the coordina-
tion of the ‘type 3 copper’ centre [5,6].
mel genes
The frequent occurrence of a melanin-negative
phenotype in S. glaucescens and Streptomyces reticuli
has been attributed to insertion elements [36,76]. In the
potato pest Streptomyces scabies and in Rhizobium
meliloti, the tyrosinase genes are plasmid located [31,61].
Genetic studies with melanin-negative Streptomyces
mutants revealed a polycistronic organization of the
chromosomal melC operon (Fig. 2). Besides the
structure gene for the tyrosinase (melC2), at least one
more upstream located gene (melC1) is needed for the
expression of the melanin phenotype in different
Streptomyces species [18,35,36,39,41–43]. The melC1
gene product is a small chaperon-like protein (Table 1)
which is involved in tyrosinase secretion and incorpora-
tion of copper (see below).
By cloning procedures, several bacterial tyrosinase
genes have been sequenced and translated into the
protein sequence [3,36,37,39,42,43,54,55,61,73,86]. In
the course of current genome projects more putative
tyrosinase genes have been identified in Gram-negative
and Gram-positive bacteria (Table 1). The molecular
masses of the corresponding proteins range between 14
and 75 kDa, those of Streptomyces are about 30 kDa. It
should be noted that putative tyrosinase genes have been
found also in Streptomyces species which are phenoty-
pically melanin-negative (e.g. S. coelicolor) and in some
genomes several tyrosinase genes have been identified
(S. avermitilis).
Sequence alignments of many pro- and eukaryotic
tyrosinases showed that the copper binding regions are
highly conserved. The signatures of CuA and CuB are H-
x(n)-H-x(8)-H and H-x(3)-H-x(n)-H, respectively. An-
other conserved histidine does not contribute to the
copper binding but is required for correct substrate
orientation at the active site [54]. The Streptomyces
tyrosinases comply with this general scheme (Fig. 3). In
addition to the conserved histidines, more invariant
regions can be found within the primary sequences which
may be useful targets for structure/function analyses.
Fig. 2. Schematic drawing of the mel operon from Streptomyces antibioticus (modified from 4). Boxes represent coding regions; open
circles: 10 and 35 promoter regions; closed circles: ribosome binding sites; arrow: transcription start.
7
Apart from these essential conserved regions, we
found significant sequence variations among bacterial
tyrosinases. Those from Gram-negative bacteria share a
20–30% homology to each other and to the Gram-
positive bacteria. Within the streptomycetes, the overall
relationship varies between 36% and 86%.
The protein from Stenotrophomonas maltophila shows
no significant sequence similarity to any other bacterial
tyrosinase. With only 169 amino acids it is the smallest
tyrosinase sequenced [86] and most interesting only two
histidines but three cysteine residues occur in the
primary sequence. Obviously, only a small protein
backbone is essential for its catalytic activity and one
may wonder about the number and binding mode of
copper atoms.
Amino acid composition
Alanine is the dominating amino acid in the
prokaryotic tyrosinase sequences. In Gram-positive
bacteria there are also high amounts of charged residues
(arginine, aspartate). Arginine residues favour not only
the formation of salt bridges, but may simultaneously
bind to aromatic stacked rings [28].
Cysteine is missing in the tyrosinases of streptomy-
cetes except in those of Streptomyces griseus and in one
of three putative tyrosinase sequences identified in the
genome of Streptomyces avermitilis. The chaperon-like
activating proteins of Streptomyces tyrosinases are, with
the exception of S. lincolnensis, also devoid of cysteine
but rich in histidines. Hence it follows that predomi-
nantly histidine and not cysteine residues contribute to
metal binding in Streptomyces tyrosinases.
The crystal structures of the plant catecholoxidase and
mollusc haemocyanin revealed that cysteines are in-
volved in the formation of a disulphide bond and a
particular cysteine forms an unusual thioether bridge
with a histidine at the CuA site (Fig. 5). It has been
discussed that this bond stabilizes the orientation of the
copper-binding histidine which is located by a loop
instead of an alpha helix in the case of arthropod
haemocyanin. It is an interesting observation that
different classes of oxygen-activating copper enzymes
contain modified amino acids either ligated to, or in close
proximity to their active sites. Some of these unusual
residues, namely HisCys, TyrCys and TyrHis moieties,
 ARTICLE IN PRESS
8 H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14

S.ga. -MTVRKNQAALTADEKRRFVAAVLELKRNGRYDEFVRTHNEFIMS---DTRTGRRGGPGH
S.ca. -MTVRKNQATLTADEKRRFVAAVLELKRSGRYDEFVRTHNEFIMS---DTDSGER--TGH
S.av.(Q93HL2) -MTVRKNQATLTADEKRRFVDALVALKRSGRYDEFVTTHNAFIMG---DTDSGER--TGH
S.gl. -MTVRKNQATLTADEKRRFVAAVLELKRSGRYDEFVTTHNAFIIG---DTDAGER--TGH
S.an. -MTVRKNQASLTAEEKRRFVAALLELKRTGRYDAFVTTHNAFILG---DTDNGER--TGH
S.li. -MTVRKNQATLTADEKRRFVTAVLSSS-AARYDTFVTTHNEFIVA---DTDNGER--TGH
S.ta. -MTVRKNQATLTADEKRRFVNALLELKRSGQYDTFVTTHNAFIMS---DTDNGDR--VGH
S.gr. MVHVRKNHLTMTAEEKRRFVHAVLEIKRRGIYDRFVKLHIQVNSTDYLDKESGKR--LGH
S.av.(Q93HK5) -MYTRKDVSTLTRSERRRLVAALLEVKRRGEYDEFVRMHIKYYVA---DGETGLR--AAH
S.co. MAYTRKDVSTLTRTEKRRFVNALLEIKRRGEYDEFVRTHIEYYVS---DGENGLR--TAH
.**: ::* *:**:* *:: . . ** ** * * * * .*

S.ga. RLPLPFLPWHRRFLLDFEQALQSVDSSVALPYWDWSTDRTVRASLWAPDFLGGTGRSSDG
S.ca. RSPS-FLPWHRRFLLDFEQALQSVDSSVTLPYWDWSADRTVRASLWAPDFLGGTGRSTDG
S.av. RSPS-FLPWHRRFLIEFEQALQAVDPSVALPYWDWSTDRTARASLWAPDFLGGSGRSLDG
S.gl. RSPS-FLPWHRRYLLEFERALQSVDASVALPYWDWSADRTARASLWAPDFLGGTGRSLDG
S.an. RSPS-FLPWHRRFLLEFERALQSVDASVALPYWDWSADRSTRSSLWAPDFLGGTGRSRDG
S.li. RSPS-FLPWHRRFLLEFERALQSVDASVALPYWDWSTDRSARSSLWAPDFLGGTGRSRNG
S.ta. RSPS-FLPWHRRFLIQFEQALQSVDATVTLPYWDWTADRTSRSSLWAPDFLGGTGRARDG
S.gr. VNPG-FLPWHRQYLLKFEQALQKVDPRVTLPYWDWTTDHGENSPLWSDTFMGGNGRPGDR
S.av. MAPS-FLPWHRMFLLDLERVLRRVDESVTMPYWDWTRSRSRTAAPWTEDLLGGTGRQSDR
S.co. MAPS-FLPWHRRFLLDLEEALRRVDPSVTVPYWDWTKDRSAKSAPWTADLLGGTGRRSDH
* ****** :*:.:*..*: ** *::*****: .: :. *: ::**.** :

S.ga. RVMDGPFAASTGNWPVNVRVDGR----TFLRRSLGTGVR--ELPTR------------AE
S.ca. RVMDGPFAAFTGNWPINVRVDSR----TYLRRSLGGSVA--ELPTR------------AE
S.av. RVMDGPFAASTGNWPVNVRVDSR----TYLRRTLGGGGR--ELPTR------------AE
S.gl RVMDGPFAASAGNWPINVRVDGR----AYLRRSLGTAVR--ELPTR------------AE
S.an. QVMDGPFAASAGNWPINVRVDGR----TFLRRALGAGVS--ELPTR------------AE
S.li RVTDGPFRAATGVWPITVRLDGR----TYLRRALGGAGR--ELPTR------------AE
S.ta. QVTDGPFARTGNRWTINVRVDGR----DYLRRDLGAGGR--QLPTR------------AE
S.gr. RVMTGPFARRNG-WKLNISVIPEGPEDPALNRQLHPRRP--RLPRTGLRHAHPGPADPAE
S.av. QVTTGPFAYRHGGWPIKEGITDA----EYLMRDLGRSRDPIALPTA------------RD
S.co. RVTTGPFAHAGGNWTIKVNVTDT----EYLTRDLGRAADPLGLPTK------------SD
:* *** . * :. : * * * ** :

S.ga. VDSVLSMATYDMAPYNSASDG------FRNHLEG-----WRG------VNLHNRVHVWVG
S.ca. VESVLAISAYDLPPYNSASEG------FRNHLEG-----WRG------VNLHNRVHVWVG
S.av. VDSVLAMSTYDMAPWNSASDG------FRNHLEG-----WRG------VNLHNRVHVWVG
S.gl. VESVLGMATYDTAPWNSASDG------FRNHLEG-----WRG------VNLHNRVHVWVG
S.an. VDSVLAMATYDMAPWNSGSDG------FRNHLEG-----WRG------VNLHNRVHVWVG
S.li. VDSVLSIPTYDMAPWNSASDG------FRNHLEG-----WRG------VNLHNRVHVWVG
S.ta. VDSVLAMETYDMAPWNSSSDG------FRNHLEG-----WRG------VNLHNRVHVWVG
S.gr. LEQTLDLTVYDCPPWNHTSGGTPPYESFRNHLEGYTKFAWEPRL----GKLHGAAHVWTG
S.av. LQGALDDPVYDTAPWNSTSAKG-----FRNRLEG-----WGPGRGAASWRNHNRVHRWVG
S.co. LEWALDDPKYDTSPYDSTVRKG-----FRNKLEG-----WGAGRGSVSWRNHNRVHRWVG
:: .* ** .*:: ***:*** * . *. .* *.*

S.ga. GQMATGVSPNDPVFWLHHAYNRQLWAEWQRRHPGA-GYVPT-------GGTPDVVDLNDT
S.ca. GQMATGVSPNDPVFWLHHAYVDKLWAEWQRRHPDS-AYVPT-------GGTPDVVDLNET
S.av. GQMATGVSPNDPVFWLHHAYIDRLWAQWQSRHPGS-GYVPT-------GGTPNVVDLNET
S.gl. GQMATGMSPNDPVFWLHNAYVDKLWAEWQRRHPGS-GYLPA-------AGTPDVVDLNDR
S.an. GQMATGVSPNDPVFWLHHAYIDKLWAEWQRRHPSS-PYLPG-------GGTPNVVDLNET
S.li. GQMATGVSPNDPVFWLHHAYIDKLWAQWQRRHRTP-AYVPA-------AGTPDVVDLDET
S.ta. GQMATGVSPNDPVFWMHHAFVDKLWADWQARHPRS-TYLPA-------AGTANVVDLGDT
S.gr. GHMMYIGSPNDPVFFLNHCMIDRCWALWQARHPDVPHYLPT-------VPTQDVPDLNTP
S.av. GHMVSGASVNDPVFWMHHAFVDLLWSRWQQRHQGA-RYLPEQQPARGDAQRGRIVARHER
S.co. GAMVGGASVNDPVFWLHHAFIDLQWSRWQARHRGA-RYLPAEPPGRGSAQRGRIVARHEK
* * * *****::::. *: ** ** *:* :

S.ga. MKPWNDVRPADLLTHTAHYTFDV--
S.ca. MKPWNTVRPADLLDHTAYYTFDA--
S.av. MKPWNDVRPADLLDHTAHYTFDTV-
S.gl. MKPWNDTSPADLLDHTAHYTFDTD-
S.an. MKPWNDTTPAALLDHTRHYTFDV--
S.li. MKPWHDSSPADLLDHTGHYTFDTD-
S.ta. MRPWNDVTPADMLDHTRHYTFDTAA
S.gr. LGPWHTKTPADLLDHTRFYTYDQ--
S.av. MPPWD-VTPDQLEDHSGIYRYA---
S.co. LPPWD-VTPDELEDVGRIYRYA---
: **. * : * :

Fig. 3. Sequence alignments of the tyrosinase (MelC2) proteins from Streptomyces species. Asterisk denotes identical residues in the
alignment (all conserved histidines are in bold); colon: conserved substitutions; period; semi-conserved substitutions. Cysteines
indicated all a frame; for species abbreviations see Table 1.
 ARTICLE IN PRESS
H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14 9

S.gl. MPELSRRR
RRRALGAAAALA--A--AAGTQAVAAPAAT-AAGHHPGPSTAATGHHPGT-
S.li. MPRLTRRR
RRRALTAAAALASGAGAGAGAQAAAAPGA--AAHDHGSPDVPLPCSL----
S.an. MPELTRRR
RRRALGAAAVVA------AGVPLVALPAARADDRGHHTPEVPGNPAASGAP
S.ca. MPEITRRR
RRRALTAAAAVAA-T--ASAAVTLAAPAASAAGHHEPAAPESFDEVYK---
S.ga. MPDITRRR
RRRAYTTAAAVAA-T--ASAAAPTAAPAATAAARHDHTAPDSFDEVYK---
S.ta. MSSITRRR
RRRALGVAAGAAG---AAAGLALAGQAVAAPRAAAPAAAPASFDEVYQ---
S.gr. MPMNRREMSCHHRGALAA----AAAVPLLSGGEGEGAAEAAAAPRSQRR
RRGRSTP--
S.co. MVGNAGATANGAEREYAT----EDPARGGPASGTRR
RRQVMRGLFAPALAVGLAPL--
S.av.(Q93HK6) MTLAPVLAASGSAGPEE-----ARFDETYRGRR
RRIVGDRYDAQRSGDAYSGAWHVT-

Fig. 4. Partial sequence alignments of the MelC1 proteins from Streptomyces species. Putative signal sequences are underlined. They
display three regions, a positive charged N-terminus, a hydrophobic core and C-terminal recognition site for the cleavage by specific
signal peptidases [64]. Arginine residues are in bold; for species abbreviations see Table 1.

are probably derived from a cross-linking reaction and extracellular, they contain like all bacterial tyrosi-
between two adjacent amino acid side chains [32]. nases studied so far, no signal sequences. However, as
shown in Fig. 4, most MelC1 proteins possess a typical
leader peptide, which is cleaved off after secretion [64].
Mutations in the signal sequences prevented the trans-
Incorporation of copper
port of both MelC1 and MelC2 proteins. These results
suggest a mechanism by which the apotyrosinase forms
The melanin operon of S. antibioticus [3,4,42], S.
a binary complex with the MelC1 protein followed by
glaucescens [36,37], Streptomyces lavendulae [43] and S.
incorporation of copper and transport across the
castaneoglobisporus [39] consists of two parts: melC1
cytoplasmic membrane [52,81]. The putative MelC1
which codes for a small helper protein and the tyrosinase
protein of Streptomyces coelicolor lacks a typical
structure gene melC2 (Fig. 2). Genetic and biochemical
cleavage site, which might explain the melanin-negative
studies predominantly with S. antibioticus, have shown
phenotype of the species. A cleavage site could be
that the MelC1 protein is responsible for incorporation
detected only in one of two putative Mel1C1 proteins
of copper and thus for activation of the apotyrosinase
(Q93HL1) identified in the genome of S. avermitilis.
[10,49]. The histidines of the activating proteins may
A critical point of this consideration is that the sec-
serve as the copper ligands: mutational exchanges of
pathway implicates the unfolding of the holoenzyme and
specific histidines in the MelC1 protein resulted in
thus loss of essential copper ions needed for enzymatic
significant losses of tyrosinase activity [11]. The MelC1
activity. The TAT pathway (twin-arginine translocation
and MelC2 proteins form stable binary complexes which
pathway) is a relatively newly discovered route which
can be purified by chromatographic methods. Addition
allows transport of (metallo)proteins in their native
of copper to the binary complexes resulted in incorpora-
folded conformation [74,75]. Proteins secreted in such
tion of two copper molecules and release of the activated
way display a characteristic twin-arginine motif between
tyrosinase [10]. Recently, a protein of 250 amino acids
the charged N-terminus and the hydrophobic core of the
has been demonstrated to be involved in copper transfer
leader peptide. The MelC1 proteins are in accordance
to the apotyrosinase of the Gram-negative M. mediter-
with that recognition signature (Fig. 4) and are most
ranea [55]. The responsible gene is on the same
likely transported by the TAT route, which is widely
transcription unit as the tyrosinase structure gene. For
used by streptomycetes [74,75].
other bacterial tyrosinases such helper proteins have not
yet been detected. As most eukaryotic tyrosinases are
larger than those of bacteria, it has been argued that here
an internal domain may be responsible for the copper
incorporation in tyrosinases [28].
Laccases
Melanization in Streptomyces and other microrgan-
isms is not only catalysed by tyrosinases but also via
Secretion different pathways and enzymes [7,8,65]. Laccases (EC
1.10.3.2, p-diphenol: dioxygen oxidoreductases) are
In prokaryotes (bacteria and archaea) transport of multi-copper proteins that use molecular oxygen to
extracellular proteins across the cytoplasma membrane oxidize various aromatic and non-aromatic compounds.
is mainly managed by the universal secretion pathway [13,77]. Until recently, laccases have been only found in
(sec-pathway) [68]. Specific signal sequences of the eukaryotes (fungi, higher plants, insects) but now there
proteins are recognized by the SRP (signal recognition is strong evidence for their wide distribution in
particle) and directed to the secretion apparatus. prokaryotes [13]. For the catalytic activity a minimum
Although Streptomyces tyrosinases are found intra- of four copper atoms (type 1, type 2, type 3) per active
 ARTICLE IN PRESS
10 H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14
protein unit is needed [13,77]. The occurrence of a spin-
coupled copper pair (type 3 copper) is the common
feature of the multi-copper oxidases and the protein
superfamily of tyrosinases and haemocyanins
[13,20–23,40,50,77].
In contrast to tyrosinases, laccases exhibit no mono-
phenolhydroxylase activity, but oxidize phenols by a
radical-generating reaction mechanism [13]. Some mi-
croorganisms show both tyrosinase and laccase activ-
ities, and care must be taken to avoid confusion because
of overlapping substrate specificities (Fig. 1). In
Sinorhizobium meliloti, a plasmid-encoded tyrosinase
and a laccase have been demonstrated [8,61]. In addition
to a typical tyrosinase, a ‘multipotent’ phenoloxidase
with both tyrosinase and laccase activities has been
purified from the marine bacterium M. mediterranea
[73]. The protein shows some extra histidine-rich
copper-binding domains that are very likely related to
its unique enzymatic properties.
The phenoxazinone synthase of S. antibioticus which
is involved in the biosynthesis of actinomycin [27] is a
multi-copper enzyme with laccase activity. In the
meanwhile, more laccases have been isolated and
characterized from Streptomyces cyaneus [1], S. griseus
[26], S. lavendulae [78] and S. coelicolor [57].
Physiological importance and applications
In spite of their ubiquitous distribution in nature and
intensive biochemical investigations, the biological func-
tions of bacterial tyrosinases are not fully understood. In
streptomycetes, tyrosine is neither best substrate nor
inducer of the tyrosinases. The best-documented func-
tion of the enzyme is restricted to the formation of
melanins. The dark pigments protect the bacterial cells
and spores against UV radiation [7,65,71,72]. For
instance, the expression of the mel gene from Pseudo-
monas maltophila significantly increased the viability of
B. thuringiensis [71]. Melanins bind heavy metals that are
otherwise toxic to the cells [7]. They also confer
protection against oxidants, heat, enzymatic hydrolysis,
antimicrobial compounds and phagocytosis and thus can
contribute to microbial pathogenesis [65]. Since melanin-
negative mutants are often affected in aerial mycelium
and spore formation, a physiological function of
tyrosinases in cell morphogenesis of Streptomyces has
been discussed. In soil environments, extracellular
tyrosinases are probably involved in the polymerization
and detoxification of plant phenolic compounds and the
formation of humic matter [14,15,47].
Monophenols and o-diphenols have been considered
as the exclusive tyrosinase substrates for a long time.
However, aromatic amines and o-aminophenols have
been also recognized as tyrosinase substrates [15,29,50]
which undergo similar ortho-hydroxylation and oxida-
tion reactions.
The monophenolhydroxylase and diphenoloxidase
activities of tyrosinase are the basis for many industrial
applications [25]: in the environmental technology for the
detoxification of phenol-containing waste waters [14],
contaminated soils [15] and as biosensors for the
monitoring of phenols; in pharmaceutical industries for
the production of o-diphenols (e.g. L-dopa, dopamine for
the treatment of Parkinson‘s desease); in cosmetic and
food industries, because of either undesirable or beneficial
oxidative browning reactions [60]. Synthetic melanins
have applications as protectives against radiation (UV,
X-ray, gamma-ray), cation exchanger, carrier for drugs,
antioxidants, antiviral agents and immunogens [65,86].
The establishment of systems for the overproduction
of the mel gene in various Gram-positive and Gram-
negative bacteria [24,45,82] enables industrial enzyme
production. Bacterial tyrosinases with new features like
high-temperature stabilities [46,53] and broader sub-
strate spectra [73] open further application areas.
Relationship to other ‘type 3 copper’ proteins
Whereas a lot of information about genes, sequences,
mutations and kinetics is available for Streptomyces
tyrosinases, not much is known about their structure.
The comparison with other ‘type 3 copper’ proteins such
as haemocyanins may help to get a funded idea
[20–23,33,40,48,66,79,80,85], Recently it was shown that
haemocyanins exhibit a tyrosinase activity after specific
activation [20,22,23,40]. Consequently, our present
knowledge of tyrosinase structure has been mainly
derived from the resolved crystal structures of haemo-
cyanins [19,33,58,66] and the catecholoxidase of a sweat
potato [30,44]. Based on a thorough comparison of
sequences and structures of haemocyanins, tyrosinases
and catecholoxidases, two different types of tyrosinases
seem to exist [20,21,23,40,50,85]. One type (a-tyrosinase)
equals arthropod haemocyanins with subunits folding
into three domains. The other type (m-tyrosinase) is
similar to the functional units of molluscan haemocya-
nin subunits, which consist of two domains. In contra-
diction to a-tyrosinase, in the case of m-tyrosinase the
C-terminal domain has to be removed before phenolic
substrates can approach the active site [20–23,40]. In
addition, one of the histidines coordinating CuA,
provided by a loop instead of an a-helix, is arrested by
an odd cystein-histidine bond, which seems typical for
m-tyrosinases (Fig. 5). Since Streptomyces tyrosinases
are usually devoid of cysteine, they may belong to the a-
tyrosinase group or a new one.
Fig. 6 shows a 3D homology model of the tyrosinase
from S. antibioticus based on the known crystal
 ARTICLE IN PRESS
H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14 11

structure of the catecholoxidase from the sweat potato

Ipomoea batatas. An open view into the active site
reveals no steric hindrance for bulky compounds such as
phenols. This simplified model encourages the expecta-
tion that bacterial tyrosinases may be promising
candidates to understand the catalytic mechanism and
activation of ‘type 3 copper’ proteins on a molecular
level. In comparison to eukaryotic cells they have some
advantages: (1) they can be rapidly cultured at high cell
densities, (2) the tyrosinases are secreted into the culture
medium, (3) the active from is most probably a small
monomer and (4) the protein is not post-translationally
modified. In addition, bacterial systems are ideal for
site-directed mutations and the natural diversity of the
Streptomyces tyrosinases may be a key for comparative
mechanistic studies.

Fig. 6. Homology modelling of Streptomyces tyrosinase.

Acknowledgements
H.C. wants to dedicate this article to Prof. Dr. H.J.
Kutzner. The modelled structure of Strepto-
myces tyrosinase was kindly provided by Thorsten
Modeller 6 was used according to [59]. The 3D structure of
the catecholoxidase from Ipomoea batatas (1BT1) was used as
a template. The view into the open active site allows to see the
two copper atoms (red), copper-liganding histidines (blue),
Asn-190 (green, important for tyrosinase activity), Gly-204
(orange).

Fig. 5. Active sites of ‘type 3 copper’ proteins based on crystal structures. The two copper atoms are coordinated by six histidines,
which are provided by a four alpha helix bundle. In the oxy-state the molecular oxygen reversibly bound as a peroxide between the
two copper atoms (Cu A and CuB) in a ‘side-on’ conformation. (A) Deoxy-form of the catecholoxidase from Ipomoea batatas
(arrow: His–Cys thioether bond) [44]; (B) oxy-form of mollusc-haemocyanin [19]; (C) oxy-form of arthropod haemocyanin [58]; (D)
enlarged superposition of the three copper centres.
 ARTICLE IN PRESS
12 H. Claus, H. Decker / Systematic and Applied Microbiology 29 (2006) 3–14
Schweikardt and Uwe Salzbrunn (Institute for Mole-
cular Biophysics, University of Mainz).
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